Development of a Multiplex PCR and Magnetic DNA Capture
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pathogens Article Development of a Multiplex PCR and Magnetic DNA Capture Assay for Detecting Six Species Pathogens of the Genera Anaplasma and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples from Grenada, West Indies Bhumika Sharma 1,* , Roman R. Ganta 2, Diana Stone 1, Andy Alhassan 1 , Marta Lanza-Perea 3, Vanessa Matthew Belmar 1, Inga Karasek 4, Elizabeth Cooksey 4, Catherine M. Butler 4, Kathryn Gibson 1,† and Melinda J. Wilkerson 1 1 Department of Pathobiology, School of Veterinary Medicine, St. George’s University, St. George, West Indies, Grenada; [email protected] (D.S.); [email protected] (A.A.); [email protected] (V.M.B.); [email protected] (K.G.); [email protected] (M.J.W.) 2 Center of Excellence of Vector Borne Diseases, Department of Diagnostic Medicine/Pathobiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA; [email protected] 3 Department of Small Animal Medicine & Surgery, School of Veterinary Medicine, St Georges University, St. George, West Indies, Grenada; [email protected] 4 Department of Large Animal Medicine & Surgery, School of Veterinary Medicine, St. George’s University, St. George, West Indies, Grenada; [email protected] (I.K.); [email protected] (E.C.); Citation: Sharma, B.; Ganta, R.R.; [email protected] (C.M.B.) Stone, D.; Alhassan, A.; Lanza-Perea, * Correspondence: [email protected] M.; Matthew Belmar, V.; Karasek, I.; † Current address: Office of Field Operations, Food Safety and Inspection Service, Cooksey, E.; Butler, C.M.; Gibson, K.; United States Department of Agriculture, Edinburg, VA 22824, USA. et al. Development of a Multiplex PCR and Magnetic DNA Capture Abstract: Infections with tick-borne pathogens belonging to Anaplasma/Ehrlichia in various vertebrate Assay for Detecting Six Species hosts are a persistent problem resulting in nonspecific clinical signs during early infection. Diagnosis Pathogens of the Genera Anaplasma of single and multi-infections with these pathogens, causing diseases in companion/agricultural and Ehrlichia in Canine, Bovine, Caprine and Ovine Blood Samples animals and people, remains a challenge. Traditional methods of diagnosis, such as microscopy and from Grenada, West Indies. Pathogens serology, have low sensitivity and specificity. Polymerase chain reaction (PCR) assays are widely 2021, 10, 192. https://doi.org/ used to detect early-phase infections, since these have high sensitivity and specificity. We report 10.3390/pathogens10020192 the development and validation of an assay involving PCR followed by magnetic capture method using species-specific oligonucleotides to detect six Anaplasma/Ehrlichia species pathogens in canine, Academic Editor: bovine, caprine, and ovine blood samples. Overall, the assay application to 455 samples detected Alejandro Cabezas-Cruz 30.1% (137/455) positives for one or more out of six screened pathogens. Single-pathogen infections Received: 30 December 2020 were observed in 94.9% (130/137) of the positive samples, while co-infections were detected in 5.1% Accepted: 7 February 2021 (7/137). Anaplasma marginale infection in cattle had the highest detection rate (34.4%), followed Published: 10 February 2021 by canines positive for Anaplasma platys (16.4%) and Ehrlichia canis (13.9%). The assay aided in documenting the first molecular evidence for A. marginale in cattle and small ruminants and Ehrlichia Publisher’s Note: MDPI stays neutral chaffeensis and Ehrlichia ewingii in dogs in the Caribbean island of Grenada. with regard to jurisdictional claims in published maps and institutional affil- Keywords: Anaplasma; Ehrlichia; PCR; xMAP iations. 1. Introduction Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. For over three decades, Anaplasma and Ehrlichia species pathogens have been known This article is an open access article to cause diseases in humans, while in pets and livestock these infections have been well- distributed under the terms and documented for many decades [1–3]. In the United States, human infections with Anaplasma conditions of the Creative Commons and Ehrlichia species are identified as the second leading cause of tick-borne diseases after Attribution (CC BY) license (https:// Lyme disease [4]. Clinical outcomes of ehrlichiosis and anaplasmosis vary from asymp- creativecommons.org/licenses/by/ tomatic infections to severe, potentially fatal illness in animals and humans. Two or more 4.0/). Pathogens 2021, 10, 192. https://doi.org/10.3390/pathogens10020192 https://www.mdpi.com/journal/pathogens Pathogens 2021, 10, 192 2 of 18 tick-borne infections are also common in vertebrate hosts [5–17]. Multi-infections with tick- borne pathogens may enhance the disease severity and complicate the clinical presentation in a host [18–20]. Gaunt et al. [21] reported a greater pathophysiological response in dogs experimentally co-infected with Ehrlichia canis and Anaplasma platys, than when infected with either one of the pathogens. Multiple-pathogen infections can also persist for months to years and complicate a patient’s clinical presentation, substantially influencing the pro- gression of the diseases, while also creating challenges for laboratory diagnosis [13,22,23]. Early detection of these infections, when antibiotic treatment is most effective, is often very challenging. This is because early signs and symptoms of these illnesses are nonspecific, making clinical diagnosis difficult [24]. Assays that can rapidly confirm and discriminate between tick-borne rickettsial pathogens are limited and not readily available at an affordable cost. The Indirect immunofluorescence antibody (IFA) assays performed on paired acute and convalescent sera are considered the gold-standard for serologic con- firmation of rickettsial infections [25,26]. However, IFA assays are insensitive during the acute phase of rickettsial infection [24,27–30], because during the early infection stage pathogen-specific antibodies are yet to develop. For tick-borne rickettsial infections, se- roconversion usually occurs within two to four weeks, at which time pathogen-specific antibodies can be detected. Therefore, the Centers for Disease Prevention and Control, USA, recommends performing an IgG IFA assay on acute and convalescent-phase samples (sampled two to four weeks apart) in tandem, a four-fold or greater increase in the antibody titer is evidence of seroconversion and reflects current infection [30]. For the detection of Anaplasma marginale in particular, the World Organization for Animal Health recom- mends performing microscopic examination of the freshly prepared blood smears as well as polymerase chain reaction (PCR) assays [31]. PCR assays are based on the principle of artificial amplification of species-specific DNA and are widely used for rapid, sensitive, and specific detection of Anaplasma/Ehrlichia species, in the whole blood specimens collected during the acute stages of illness. These molecular methods include both conventional and real-time quantitative PCR assays targeting mostly 16S rRNA or 16S rDNA [6,32–37]. Other PCR assays use primers targeting genes such as dsb [38], groEL [39,40], msp1a, and msp4 of A. marginale [41,42], major outer membrane protein genes of Ehrlichia species such as p28-p30/MAP1 [43,44], and citrate synthase gene gltA [45]. However, most of these assays only detect a limited number of Anaplasma/Ehrlichia species. Recently, new technologies have been developed for molecular diagnosis of tick-borne rickettsial infections and identi- fying the infecting agent. For example, Michelet et al., [46] utilized a microfluidics system to perform parallel real-time PCRs to test ticks for the presence of 25 bacterial and 12 parasitic species simultaneously. In Grenada, one of the Windward Islands of the Caribbean, Anaplasma and Ehrlichia infections are highly prevalent in dogs, and are considered endemic in cattle infections with A. marginale as judged by serological analysis [47–53], and have recently been reported in small ruminants [54]. In dogs, these infections are primarily transmitted by the vector Rhipicephalus sanguineus (brown dog tick). Reports on PCR and serology-based assays have identified co-infections in dogs of Grenada to both E. canis and A. platys [48–50]. Molecular evidence of Anaplasma and Ehrlichia infections in ruminants in Grenada, however, is lim- ited, and infections by multiple pathogens are also not well documented [47]. Moreover, Ehrlichia ewingii, Ehrlichia chaffeensis, and Ehrlichia ruminantium infections have not previ- ously been reported from Grenada, although they have been documented from some of the islands of the Caribbean [47–50,54,55]. Grenada experiences both human and animal international movements throughout the year from the Americas, Africa, Asia, and Europe since it is an educational hub and a popular tourist destination. With the population influx, the possibility of the introduction of exotic ticks and tick-borne rickettsial pathogens in Grenada is high. The currently available assays in Grenada (point-of-care enzyme-linked immunosorbent assay and conventional PCR) only focus on E. canis and A. platys due to the endemic status of these two pathogens. Due to globalization and Grenada’s unique Pathogens 2021, 10, 192 3 of 18 status as a tourist and educational destination, this is no longer sufficient for the detection and control of tick-borne rickettsial infection. Therefore, in this study, we report the development and validation of a new multiplex PCR coupled with oligonucleotide probe based multi-analyte profiling (xMAP)